A fast, efficient chromatin immunoprecipitation method for studyingprotein-DNA binding in Arabidopsis mesophyll protoplasts

Collection with item attached
2017
Item details URL
http://open-repository.kisti.re.kr/cube/handle/open_repository/473588.do
DOI
10.1186/s13007-017-0192-4
Title
A fast, efficient chromatin immunoprecipitation method for studyingprotein-DNA binding in Arabidopsis mesophyll protoplasts
Description
The work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government (2017R1A2B3009624 to J.H.A.),Basic Science Research Program through the National Research Foundationof Korea (NRF) grant funded by the Ministry of Education(2015R1D1A4A0101941 to J.H.L.).
abstract
Background: Binding of transcription factors to their target sequences is a primary step in the regulation of gene expression and largely determines gene regulatory networks. Chromatin immunoprecipitation (ChIP) is an indispensable tool used to investigate the binding of DNA-binding proteins (e.g., transcription factors) to their target sequences in vivo. ChIP assays require specific antibodies that recognize endogenous target transcription factors; however, in most cases, such specific antibodies are unavailable. To overcome this problem, many ChIP assays use transgenic plants that express epitope-tagged transcription factors and immunoprecipitate the protein with a tag-specific antibody. However, generating transgenic plants that stably express epitope-tagged proteins is difficult and time-consuming.
Results: Here, we present a rapid, efficient ChIP protocol using transient expression in Arabidopsis mesophyll protoplasts that can be completed in 4 days. We provide optimized experimental conditions, including the amount of transfected DNA and the number of protoplasts. We also show that the efficiency of our ChIP protocol using protoplasts is comparable to that obtained using transgenic Arabidopsis plants. We propose that our ChIP method can be used to analyze in vivo interactions between tissue-specific transcription factors and their target sequences, to test the effect of genotype on the binding of a transcription factor within a protein complex to its target sequences, and to measure temperature-dependent binding of a transcription factor to its target sequence.
Conclusions: The rapid and simple nature of our ChIP assay using Arabidopsis mesophyll protoplasts facilitates the investigation of in vivo interactions between transcription factors and their target genes.
provenance
Made available in Cube on 2018-09-28T10:23:38Z (GMT). No. of bitstreams: 0
language
English
author
Lee, Jeong Hwan
Jin, Suhyun
Kim, Sun Young
Kim, Wanhui
Ahn, Ji Hoon
accessioned
2018-09-28T10:23:38Z
available
2018-09-28T10:23:38Z
issued
2017
citation
PLANT METHODS(13)
issn
1746-4811
uri
http://open-repository.kisti.re.kr/cube/handle/open_repository/473588.do
Funder
교육부
Funding Program
BK21플러스사업(0.5)
Project ID
1345273981
Jurisdiction
Rep.of Korea
Project Name
KU Advanced Graduate Program for Life Science
rights
openAccess
subject
Arabidopsis
Chromatin immunoprecipitation
Protoplasts
Transcriptionfactor
Transient expression
type
article


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